The overall goal of this proposal is to identify the Ras isoforms important for the pathogenesis of malignant peripheral nerve sheath tumors (MPNSTs) and to determine whether these isoforms are activated in response to stimulation by the growth factor NRG-1. The first specific aim will test the hypothesis that NRG- 1 promotes MPNST mitogenesis and migration by activating specific members of the classic Ras and R-Ras subfamilies. The second specific aim will test the hypothesis that these same molecules are important for MPNST tumorigesis in vivo. The third specific aim will test the hypothesis that the Ras isoforms important for MPNST proliferation and migration are activated by specific guanine nucleotide exchange factors (GEFs) in response to NRG-1 signaling. The rationale for these investigations stems from recent studies in which individual Ras isoforms have been shown to have unique functions and varying responses to therapeutic agents, emphasizing the importance of determining which Ras isoforms are relevant to the pathogenesis of a particular tumor type prior to designing therapies to inhibit Ras signaling in that tumor. In the first specific aim, dominant negative mutants and short hairpin RNAs will be used to inhibit the activity of Ras isoforms of the classic Ras (H, N, and K-Ras) and the R-Ras (R-Ras, R-Ras2, M-Ras) subfamilies, and the effects of this inhibition on proliferation, migration, and colony formation will be determined in the unstimulated and NRG-1 stimulated states. Unstimulated and NRG-1 stimulated cells will also be assayed for Ras activation to determine which Ras isoforms are NRG-1 responsive. In the second specific aim, the contribution of these isoforms to tumorigenesis in vivo will be determined by non-invasive imaging of tumor growth in immunodeficient mice following sciatic nerve grafting of MPNST cells stably expressing shRNAs directed against specific Ras isoforms. In the third specific aim, short hairpin RNAs will be used to ablate the expression of specific GEF proteins, and the effects of this ablation of Ras activation, colony formation, proliferation, and migration will be assessed in the presence and absence of NRG-1 stimulation to determine the specific mechanisms by which NRG-1 is capable of activating Ras proteins. Relevance to Public Heath: Malignant peripheral nerve sheath tumors are the most common malignancy that develops in patients with neurofibromatosis type I. Surgery is the primary treatment for these tumors, and there are few therapeutic options available if complete surgical resection is not obtained. This work attempts to better define the cellular signaling pathways necessary for MPNST proliferation and migration in order to identify potential targets for chemotherapeutic intervention.